The biggest challenges are locating effective, reasonably priced, and eco-friendly compounds to treat diseases caused by insects and microbes. The aim of this study was to employ GC-MS to assess the biological potency and chemical composition of the aerial parts of Reichardia tingitana (L.) Roth. Using this technique, 17 components were interpreted from the extracted plant, accounting for around 100% of total volatile compounds. Commonly, 6,10,14-trimethylpentadecan-2-one (21.98%) and methyl oleate (27.26%) were positioned as the major components, which were ascertained after 19.25, and 23.34 min, respectively. The major components were classified as hydrocarbons (23.82%), fatty acids, esters of fatty acids (57.46%), steroids (17.26%), and terpenes (1.48%). The DPPH antioxidant activity of the R. tingitana extracted components revealed that the shoot extract is the most powerful, with an IC50 value of 30.77 mg L−1 and a radical scavenging activity percentage of 71.91%. According to the current result, methanolic extract of R. tingitana had the maximum zone of inhibition against Salmonella typhimurium and Bacillus cereus (25.71 ± 1.63 and 24.42 ± 0.81 mm, respectively), while Clostridium tetani and Staphylococcus xylosus were the main resistant species. In addition, the 50% methanol crude shoot extract of R. tingitana showed greater potential anticancer activity with high cytotoxicity for two tumor cells HepG-2 and PC3 cells (IC50 = 29.977 and 40.479 µg mL−1, respectively) and noncytotoxic activity for WI-38 normal cells (IC50 = >100 µg mL−1). The MeOH extract of plant sample was more effective against Aedes aegypti larvae with LC50 of extract being 46.85, 35.75, and 29.38 mg L−1, whereas the LC90 is 82.66, 63.82, and 53.30 mg L−1 for the various time periods of 24, 48, and 72 h, respectively. R. tingitana is a possible biologically active plant. Future study will include pure chemical isolation and individual component bioactivity evaluation.